Extraction and analysis of soluble inositol polyphosphates from yeast

被引：91
|
作者：
Azevedo, Cristina
Saiardi, Adolfo
机构：
[1] UCL, MRC, Cell Biol Unit, London WC1E 6BT, England
[2] UCL, Dept Biochem & Mol Biol, Mol Cell Biol Lab, London WC1E 6BT, England
来源：
NATURE PROTOCOLS | 2006年 / 1卷 / 05期
基金：
英国医学研究理事会;
关键词：
D O I：
10.1038/nprot.2006.337
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
Soluble inositol polyphosphates are implicated in the regulation of many important cellular functions. This protocol to extract and separate inositol polyphosphates from Saccharomyces cerevisiae is divided into three steps: labeling of yeast, extraction of soluble inositol polyphosphates and chromatographic separation. Yeast cells are incubated with tritiated inositol, which is taken up and metabolized into different phosphorylated forms. Soluble inositol polyphosphates are then acid-extracted and fractionated by high-performance liquid chromatography. The radioactivity of each fraction is determined by scintillation counting. This highly sensitive and reproducible method allows the accurate detection of subtle changes in the inositol polyphosphate profile and takes less than 48 h. It can easily be applied to other systems and we have included two adaptations of the protocol, one optimized for mammalian cells and the other for Arabidopsis thaliana.
引用
收藏
页码：2416 / 2422
页数：7
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